Electrospray ionization mass spectrometry (ESI/MS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), tandem mass spectrometry (MS/MS), matrix-assisted laser desorption ionization (MALDI) mass spectrometry, accurate mass measurement for elemental composition determination, and combined capillary electrophoresis (CE) and mass spectrometry remain the techniques of current interest. MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix shows some promise for the rapid analysis of small molecules (molecular weight range = 200-1000). The ions from the low-molecular-weight matrices used for conventional MALDI/MS often obscure the low mass range of the mass spectrum because they are in 100- to 1000-fold excess to the sample. LDI mass spectra could be obtained from a wide variety of molecular structures including those compounds that would not be expected to readily absorb energy from a UV laser. Studies are ongoing to compare LDI with conventional MALDI/MS in order to define the advantages and limits of each for small molecule analysis. The goal is to choose the MALDI or LDI method that gives the best chance of providing unambiguous molecular weight information. Both normal and unconventional (e.g. graphite) MALDI matrices are being investigated in this study. LDI/MS spectra usually exhibit extensive alkali metal ion cationization and this can make determination of the molecular weight problematic, especially if the sample is impure or is a mixture. MALDI spectra obtained using graphite from pencil lead also show the same behavior in addition to substantial variation depending on the source of the pencil lead. The best overall small molecule MALDI mass spectra are obtained using 3,4-dihydroxybenzoic acid (DHB) as a matrix. The off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) is also under investigation as an alternate approach for the targeted analysis of complex biological and synthetic mixtures. This approach involves automatically collecting and spotting the HPLC eluent along with an appropriate matrix on a MALDI sample plate for subsequent MS analysis, which can include accurate mass measurement and post-source decay fragmentation analysis or MS/MS. An advantage of this approach over on-line LC/MS is the generation of an archive of the original separated sample for re-analysis. Synthetic derivatives built on a constrained diacylated glycerol scaffold (DAG-lactones) are potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents corresponding to the acyl groups R1 and R2, these DAG-lactones exhibit varying degrees of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic libraries be rapidly characterized and their structures established before biological evaluation. One method used for the initial characterization of these synthetic DAG-lactone libraries employs a combine-and-analyze strategy with rapid analysis of the resultant simple mixtures by fast atom bombardment (FAB) mass spectrometry. This approach substantially reduces the number of samples to be examined, results in enhanced analytical turn-around while providing extensive structural and sample information. It is also amenable to rapid accurate mass measurement to determine elemental composition and provide an additional dimension of structure information. ESI/MS is not suitable for analysis of these DAG-lactones because the high lipophilicity and limited basicity of these compounds results in very poor and variable ionization. Flow-injection APCI/MS of individual DAG-lactones produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach, especially if MS/MS techniques are used, appears to be complementary to FAB/MS in terms of spectral information, although it is not suitable for mixture analysis. However, flow-injection APCI/MS is amenable to automation and allows multiple analyses of the same sample to be carried out unattended. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries although variable alkali metal ion cationization complicates spectral interpretation. Optimization of matrix (see above) and automation of the sample deposition process are under investigation as an approach to resolve this problem and provide an even more rapid analysis approach. Our goal is the comprehensive structural characterization of all 96 library components in one day. Nucleoside and nucleotide prodrugs of the orally active DNA methyltransferase (DNMT) inhibitor 2(1H)-pyrimidinone riboside (zebularine) continue under investigation. Zebularine exhibits high activity in inhibiting DNA methylation both in vitro and in vivo but low potency relative to other known inhibitors of DNMT. It is postulated that incorporation of zebularine into DNA is required before the drug can function as an inhibitor of DNMT by formation of a tight complex between it and the enzyme. We have previously shown that the intracellular metabolic activation of zebularine is both complex and inefficient, resulting in only very limited DNA incorporation. Since DNA incorporation requires the activated 2'-deoxynucleotide, the metabolic bottleneck for DNA incorporation appears to be the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by ribonucleotide reductase. 2'-Deoxyzebularine itself shows no inhibitory activity for DNA methylation. We have found that this lack of activity for 2'-deoxyzebularine is due to it being an extremely poor substrate for the mammalian cellular kinases responsible for the initial phosphorylation step. A second problem with zebularine has been the rapid in vivo metabolism of the parent drug by aldehyde oxidase before it can enter cells and be phosphorylated. Nucleoside prodrugs, which are resistant to aldehyde oxidase and which can be activated by plasma esterases, are being synthesized in the LMC in an attempt to increase intracellular concentrations of zebularine and hence overall metabolic activation. Chromatographic and mass spectral methods are being used to assist the synthesis of these prodrugs and to evaluate their chemical and enzymatic stability in vitro. As a proof of principle, analysis of extracts of treated cells by CE and MS will be used to evaluate whether there is any enhancement of the production of phosphorylated metabolites.